Mcmp24h48

Spleen from 8 healthy wild-type mice of 2.5-month age was minced, filtered through a 40 μm mesh, washed with ice-cold PBS, and then suspended in 5 mL of red blood cell lysis solution (sc-296258, Santa Cruz) on ice for 5 min and then washed with PBS. Splenic T lymphocytes were cultured in RPMI medium containing 10% FBS and activated using anti-CD3 (553057, BD Biosciences) and anti-CD28 (553294, BD Biosciences) antibodies (1 μg/mL) for 24 h. Activated splenic T lymphocytes were counted and seeded (5 × 10⁵ cells/well) into the of transwell upper chambers (pore size 5 μm; Millipore, MCMP24H48), which were placed in 24-well plates containing 600 μL conditioned medium from KPPC cancer cells or KPPC;Col1^pdxKO cancer cells, or serum-free RPMI medium. T cells in the upper chambers were treated with CXCL16 neutralizing antibody (MAB503, R&D Systems, Clone #142417). After 24 hours of incubation, migrated splenic T lymphocytes, that migrated to the bottom chambers of 24-well plates, were centrifuged at 800g for 5 min, fixed with PFA, and stained with crystal violet. After PBS washes (30 min, thrice), crystal violet was dissolved by DMSO and measured at OD 590 nm using a plate reader.

Mouse RAW264.7 macrophages (2 × 10⁴ cells, suspended in 100 μL serum-free medium) were seeded into the of transwell upper chambers (pore size 5 μm; Millipore, MCMP24H48), which were placed in 24-well plates containing 600 μL conditioned medium from KPPC cancer cells or KPPC;Col1^pdxKO cancer cells, or serum-free medium. Macrophages in the upper chambers were treated with CXCR2 inhibitor SB225002 (ab120895, Abcam) or CXCL5 neutralizing antibody (NBP2–22026, Novus Biological, clone 32B3). After incubation of 12 hours, macrophages were fixed by PFA and stained with 0.05% crystal violet. The migrated macrophages (that attached to the lower surface of transwell membrane) were counted under the microscope after the removal of remaining macrophages on the upper surface of transwell membrane. For the generation of conditioned medium from cancer cells, KPPC cancer cells or KPPC;Col1^pdxKO cancer cells in T225 flasks were washed with PBS twice and incubated with serum-free RPMI medium for 24 hours. Conditioned medium collection was conducted by removing cellular components using centrifugation at 800g for 5 min and then 2000g for 10 min.